WO2012159044A1 - Mobilisation des cellules souches, réparation et régénération tissulaire - Google Patents

Mobilisation des cellules souches, réparation et régénération tissulaire Download PDF

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WO2012159044A1
WO2012159044A1 PCT/US2012/038610 US2012038610W WO2012159044A1 WO 2012159044 A1 WO2012159044 A1 WO 2012159044A1 US 2012038610 W US2012038610 W US 2012038610W WO 2012159044 A1 WO2012159044 A1 WO 2012159044A1
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compound
cells
mice
composition
blood
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PCT/US2012/038610
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Irena Tsyrlova
Fawn PETTY
Reid Von Borstel
Julian READING
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Wellstat Therapeutics Corporation
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Priority to US14/118,313 priority Critical patent/US20140088006A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids

Definitions

  • HSC hematopoietic stem cells
  • G-CSF granulocyte colony stimulating factor
  • G-CSF acts on mature bone marrow cells; cells release proteases cleaving the adhesion factors responsible for the retention of cells in bone marrow.
  • the SDF1-CXCR4 axis important for the retention of cells expressing CXCR4 in the bone marrow, is also involved in the G-CSF effect (Lapidot and Petit 2002).
  • AMD3100 a CXCR4 inhibitor, approved recently for stem cell mobilization induces a more specific mobilization of cells into the circulation than G-CSF via disruption of the CXCR4- SDF1 interaction of bone marrow cells with their microenvironment.
  • HSC can be harvested and expanded or sorted ex vivo for promoting regeneration of tissues, and especially for enhancing revascularization of ischemic tissues.
  • a preferable approach would be to use a drug that selectively mobilizes endogenous HSC and other repair-promoting progenitors from the bone marrow to enhance or enable tissue repair and revasculaization.
  • This invention provides a method of mobilizing stem cells from bone marrow of a subject, comprising administering to the subject an amount of Phe-Pro-His-Phe-Asp- Leu-Ser-His-Gly-Ser-Ala-Gln-Val (SEQ ID NO: 1) (also referred to as Compound X, or Cpd. X) effective to mobilize the stem cells.
  • This invention provides a compound (SEQ ID NO: 1) for use in mobilizing stem cells from bone marrow of a subject.
  • This invention provides the use of a compound (SEQ ID NO: 1) in the manufacture of a medicament for mobilizing stem cells from bone marrow of a subject.
  • this invention provides a pharmaceutical composition comprising a compound (SEQ ID NO: 1) for mobilizing stem cells from bone marrow of a subject.
  • This method, compound, use, and pharmaceutical composition is useful for promoting one or more of preservation, repair, or regeneration of bodily tissue, or
  • the mobilized stem cells can be collected for transplant.
  • Figure 1 Stem cell mobilization after single subcutaneous injection of Compound X.
  • Figure 2 Stem cell mobilization after single subcutaneous injection of Compound X or AMD3100.
  • Figure 3 CFU-GEMM number in peripheral blood of mice lhr after injection of Compound X daily for 4 days.
  • Figure 4 CFU-GM number in peripheral blood of mice lhr after injection of Compound X daily for 4 days.
  • Figure 5 CFU-GM and CFU-GEMM number in peripheral blood of mice injected with G-CSF and Compound X daily for 4 days.
  • FIG. 6 Cell migration toward SDFla : CD34+ cells were expanded in presence of Compound X or expanded without Compound X and pulsed for 3hr with Compound X before the assay
  • Figure 7 Cell migration toward SDFla: effect of AMD3100 in presence or absence of Compound X.
  • Figure 9 Pancreatic insulin 12 weeks after STZ treatment of C57B16 mice.
  • Figure 10 Mobilization of CFU-GEMM cells into peripheral blood of diabetic db/db mice after intravenous injection of 5mg/kg Compound X.
  • Figure 11 Wound healing rates in db/db mice treated intravenously with Compound X for 4 days.
  • Figure 12 Number of blood vessels in wounds of mice treated with Compound X versus saline.
  • Figure 13 Number of blood vessels counted in the wound dermis area on day 10 post-wounding visualized by rabbit anti-a -smooth muscle actin antibodies.
  • Figure 14 Glucose level in blood of mice before wound healing study and 7 weeks after the treatment.
  • Figure 15 Wound healing rates in db/db mice treated intravenously with Compound X for 4 days.
  • Stem cells are mobilized from the bone marrow by a stem cell- stimulatory amount of (SEQ ID NO:l).
  • the appropriate amount of (SEQ ID NO: 1) is generally from about 100 micrograms to about 10 milligrams per administration, administered from 1 time per week to 3 times per day.
  • For mobilization of stem cells in intensive acute therapy such as shortly after a stroke from 2 milligrams to 10 milligrams per administration, administered 1, 2, or 3 times per day is preferred.
  • For repeated low dose administration such as for vascular maintenance from 100 micrograms to 1 milligram per administration, administered from once per day to once per week, is preferred.
  • the appropriate amount of (SEQ ID NO: 1) is from about 100 micrograms to about 1 milligram per day for one or more days.
  • the amount can be administered on each of four consecutive days. Typical amounts are, for example, 100 micrograms, 300 micrograms, 500 micrograms, or 1 milligram per day. Thus, in more specific embodiments of this invention, the ranges are from 90 to 110 micrograms, from 280 to 320 micrograms, from 450 to 550 micrograms, or from 900 to 1100 micrograms per day. (If too little is administered, the stem cells may be inhibited rather than stimulated, and mobilization may not occur. Inhibitory doses to be avoided are typically in the range of 50 ng to 1 microgram per day in a human.) Injection, for example intravenous, subcutaneous or intramuscular injection, is a preferred route of administration.
  • Intravenous injection is most preferred for strength of response, and is generally preferred in diabetic patients because of the compromised vasculature in that population.
  • bodily tissues generally can be regenerated or revascularized.
  • pancreatic tissue is regenerated or preserved, particularly insulin-producing islets.
  • dermal tissue is regenerated, for example in wound healing.
  • a general activity that applies to repair or preservation of many different tissues is promotion or enhancement of revascularization.
  • compounds and methods of the invention are used to promote revascularization of ischemic tissues, including skeletal muscle, heart and brain.
  • compositions and methods of the invention are used to prevent pancreatic islet failure, e.g.
  • stem cells have the unique characteristic of being able to divide and to give rise to more differentiated progenitor cells ("differentiation") as well as to other stem cells ("self-renewal"). The ability to self -renew ensures that the population of stem cells is not depleted. Rapidly renewing tissues where stem cells have classically been demonstrated include hematopoietic tissue, skin, stomach, intestine, and testes.
  • Stem cells may be classified according to their differentiation potential as totipotent, pluripotent or multipotent.
  • Totipotent stem cells are capable of forming any tissue in the body. The best example of this is the fertilized egg, which gives rise to both the embryo proper as well as the placenta and supporting tissues.
  • Pluripotent stem cells can form a large subset of body tissues that can include most or all the tissues in the adult whereas multipotent stem cells have a more restricted repertoire of differentiation.
  • Tissue progenitor cells are stem cells that can only differentiate into the cells that constitute a particular type of tissue.
  • Stem cells can produce new cells to repair damage to tissues and therefore have great potential for regenerative medicine. However, they exist in small quantities in tissues and especially in peripheral blood, making it difficult to collect them or use them clinically. To increase percentage of stem cells and their progenitors in peripheral blood, their mobilization by G-CSF prior to harvest has been used extensively.
  • Mobilized stem cells can repair tissues if their homing and engraftment functions are not impaired (Rafii &Lyden, 2003). There is a need, therefore, to identify compounds that regulate mobilization of pluripotent stem cells and methods of uses for therapeutic purposes.
  • WO 97/36922 Pro-Neuron, Inc.
  • WO 96/10634 Pro-Neuron, Inc.
  • SEQ ID NO: 1 discloses a tridecapeptide with the sequence Phe- Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val (SEQ ID NO: 1), also referred to herein as 'Compound X', that acts on stem cells.
  • Compound X mobilizing activity is now demonstrated and compared to other known mobilizing agents.
  • AMD3100 a specific antagonist of SDF-1/CXCL12 binding to CXCR4, has been clinically tested and approved for synergizing with granulocyte colony-stimulating factor (G-CSF) to greatly enhance G-CSF-induced mobilization of HSCs/HPCs (Lapidot, T. and I. Petit. 2002, Lapidot et al., 2005).
  • G-CSF granulocyte colony-stimulating factor
  • CD26 is a cell-surface protein, which is a dipeptidylpeptidase IV (DPPIV) and has the capacity to truncate SDF-1/CXCL ( De Meester et al.,1999).
  • DPPIV dipeptidylpeptidase IV
  • Human DPPIV is a l lO kDa cell surface molecule it contains intrinsic dipeptidyl peptidase IV activity, which selectively removes N-terminal dipeptide from peptides with proline or alanine in the third amino acid position. It interacts with various extracellular molecules and is also involved in intracellular signal transduction cascades.
  • human DPPIV has a short cytoplasmatic domain from amino acid position 1 to 6, a transmembrane region from amino acid position 7 to 28, and an extracellular domain from amino acid position 29 to 766 with intrinsic DPPIV activity.
  • DPPIV-deficient mice exhibit resistance to diet-induced obesity, reduced fat accumulation, decreased plasma levels of leptin, increased pain sensitivity, reduced stress-like responses.
  • DPPIV has been implicated in the control of lymphocyte and immune function, cell migration, viral entry, cancer metastasis, and inflammation; deletion of CD26 resulted in decreased mobilization of HPCs in response to exogenous administration of G-CSF (reviewed in Broxmeier et al.2007). DPPIV also regulates migration of human cord blood CD34+ progenitor cells and the homing and engraftment of hematopoetic stem cells. Inhibition of DPP-4 enzymatic activity promotes human hematopoetic stem cell migration and bone marrow engraftment via potentiation of the levels of intact CXCL12/SDF-la, a physiological substrate for DPP-4 activity (Christopherson e.a 2002 and 2003).
  • EXAMPLE 1 Mobilization of Mouse Stem Cells from bone marrow into peripheral blood after single subcutaneous injection of Compound X.
  • Stimulatory doses of Compound X activate quiescent stem cells, induce them to proliferate and egress from bone marrow into peripheral blood.
  • This example shows that an increase in number of hematopoietic stem cells (HSC) and their hematopoietic progenitors (HPC) happens within lhr after injection of stimulatory doses of
  • mice C57B16 male mice, from 8weeks to 4 month of age, from Harlan were used. Mice received subcutaneous injections of saline, Compound X (50 ⁇ g/kg) or Compound X (250 ⁇ g/kg). After 1 and 4 hours, mice were anesthetized and blood was collected through the orbital sinus with heparinized capillary tubes. 20 ⁇ of blood was removed from each tube for WBC counts;
  • AMD3100 also has fast mobilizing effect and works via Stromal-Derived Factor- 1/CXCL12 mediated migration and homing. Comparison of mobilizing effect of a single dose of Compound X and an optimal published dose of AMD3100 is presented in Tables 4 and 5, and Figure 2.
  • C57B16 mice received subcutaneous injections of PBS, Compound X (250 ⁇ g/kg) or AMD3100 (5 mg/kg). After lhr blood was collected through the orbital sinus with heparinized capillary tubes. Blood from each group was pooled, counted and mononuclear cells were isolated by density gradient separation. Cells were washed, counted and plated at 100,000 per dish (5 dishes/group) for CFU- GEMM and CFU-GM colonies. CFU-GEMM colonies were scored on day 14, while CFU-GM colonies on day 7 after plating in MC3434 (Stem cell Technology).
  • the blood was stained with antibodies and a phenotype determined by FACS as described before.
  • Ficolled blood was also stained to monitor the Lin-/Sca+ phenotype.
  • the cells were washed with 3 ml FACS buffer, resuspended in 300 ⁇ FACS buffer and kept on ice, protected from light until acquired on the FACSCalibur Table 4.
  • Compound X and AMD3100 are fast mobilizers, they had different effect on bone marrow cells. Contrary to AMD3100, Compound X did not increase mobilization of immature precursor cells (Lin-) and the effect of Compound X was specific to the mobilization of CFU-GEMM and did not induce mobilization of CFU- GM.
  • EXAMPLE 2 Mobilization of Mouse Stem Cells from bone marrow into peripheral blood after multiple subcutaneous injection of Compound X.
  • the low levels of circulating HSPC are drastically increased in response to repeated stimulation with the cytokine G-CSF.
  • This example is based on a protocol for a 4-day course of once-daily Compound X injections into C57B1/6 male mice similar to G-CSF. Two effective dose levels of Compound X were found previously to mobilize HSC lhr after single injection and were selected for this experiment.
  • mice Four-month old C57B1/6 male mice (Jackson Laboratories) were subcutaneously injected with 0.9% saline, 125 ⁇ g/kg Compound X or 250 ⁇ g/kg Compound X on days 1, 2, 3 and 4 as described in Table 4. Each day, one-hour post injections, peripheral blood from 3 mice per group was harvested from the orbital plexus into EDTA- containing tubes. On day 4, a femur and spleen were also removed from mice to assess by FACS whether any toxicity aroused from repeated injections of Compound X.
  • Table 8 White blood cell number in peripheral blood of mice lhr after injection of Compound X daily for 4 days.
  • Compound X like in the previous examples did not affect mobilization of granulocytic and macrophage precursors CFU-GM.
  • EXAMPLE 3 Combined effect of multiple subcutaneous injections of Compound X with G-CSF mobilizing HSC and HPC into peripheral blood.
  • Compound X and Neupogen were diluted in PBS.
  • Mice received subcutaneous injections of PBS, Compound X (5 ⁇ g/mouse), G-CSF (5 ⁇ g/mouse) or Compound X (5 ⁇ g/mouse) + G-CSF (5 ⁇ g/mouse) for four consecutive days.
  • On the last day of injections lhour post injections, blood was collected through the orbital sinus with heparinized capillary tubes. Blood was pooled from 3 mice/group and white blood cell counts were obtained. Mononuclear cells were isolated by density gradient separation from pooled blood.
  • Table 10 White blood cell count in peripheral blood of mice injected with G-CSF and Compound X daily for 4 days.
  • G-CSF mobilized many different hematopoietic progenitors similar to the literature results, even after 8 days from the first subcutaneous injection the number of circulating
  • HSC and HPC remained high (Tables 11 and 12; Figure 5).
  • Injections of Compound X selectively increased only CFU-GEMM number as measured lhr after the last injection 1.7 times; there was no difference found at day 8 in number of CFU-GEMM or CFU- GM.
  • Compound X injected into mice that received G-CSF changed the effect of G- CSF by increasing proportion of immature CFU-GEMM and decreasing CFU-GM (Tables 11 and 12; Figure 5).
  • HSCs disappear from circulation 1-5 minutes after
  • G-CSF was shown to induce stem cell mobilization by up-regulating CXCR4 and decreasing bone marrow SDFloc; AMD3100 works via CXCR4 inhibition, thus, both G-CSF and AMD3100 (plerixafor) mobilize stem cells by disrupting the
  • SDF1/CXCR4 axis (Lapidot and Petit,2002).
  • the SDF1/CXCR4 axis was shown to have primary importance for stem cell interaction with hematopoietic niche including homing and retention of stem cells.
  • Combined effect of Compound X with G-CSF on stem cell mobilization was not synergistic like the effect of G-CSF with AMD3100, rather Compound X increased proportion of HSC among mobilized by G-CSF myelocytes, demonstrating a different mechanism of action.
  • the CD34 + population of cells from human umbilical cord blood was obtained from Lonza, defrosted and expanded ex vivo with cytokine cocktail for 6 days according to standard procedures. Briefly, 10 wells of two 12 well dishes were seeded at a density of 8xl0 4 cells per well in 2 mL of CellGenix serum free growth medium (SCGM). Cells were allowed to expand in a 37 °C incubator (5% C02, 86% RH) for 6 days. During the expansion, the 10 wells of one of the two plates received treatments of Compound X to a final concentration of 5 ng/mL (10 ⁇ L ⁇ from freshly prepared 1 ⁇ g/mL stock in SCGM) at 24 hours and 72 hours after initiation of the culture. 3 hours prior to the start of the migration assay, both control and Compound X expanded cells were pulsed with either 0, 5 ng/mL or 5 ⁇ g/mL Compound X.
  • SCGM CellGenix serum free growth medium
  • Migration assay was performed according to the method described (Tavor et al.2005). Cells were centrifuged for 5 minutes at 1100 RPM and resuspended at a density of 2xl0 6 cells/mL in CellGenix SCGM serum free medium containing 1 % Fraction V BSA either in the presence or absence of 5 ng/mL or 5 ⁇ g/mL Compound X for 3 hours.
  • the feeder tray (bottom) was prepared with 600 ⁇ L ⁇ of serum free SCGM supplemented with 1% BSA in the presence or absence of SDF-loc. All treatments were conducted in triplicate.
  • the wells of the migration chamber received 100 ⁇ L ⁇ of cells from the 2xl0 6 cells/mL suspensions, preincubated with 5 ng/mL or 5 ⁇ g/mL Compound X or Control. All treatments were conducted in triplicates.
  • the plates were placed in a 37 °C incubator (5% C0 2 , 86% RH) and cells were allowed to migrate for 4 hours. After the incubation the Transwell inserts were removed and discarded from the chamber. The cells that migrated to the lower chamber were transferred to
  • cell lysis buffer/dye solution was prepared fresh by mixing CyQuant GR dye (400X stock) with IX lysis buffer. 200 ⁇ L ⁇ of the dye/lysis solution was added to the tubes containing the migrated cells, vortexed and incubated 15 minutes at room temperature (dark). 150 ⁇ L ⁇ of the resulting cell lysates were transferred to a new 96 well plate and the
  • the migration chamber (top) received 100 ⁇ L ⁇ of cells from the 2xl0 6 cells/mL suspension, in the presence or absence of 5 ng/mL Compound X, 10 ⁇ g/mL antibodies to CXCR4 or 10 ⁇ g/mL AMD3100, as indicated.
  • 500 aliquots of control or Compound X pulsed cells were treated with 5 ⁇ L ⁇ of anti-CXCR4 (1 mg/mL in PBS) or 5 ⁇ .
  • AMD3100 (1 mg/mL in SCGM) prior to adding cells to the migration chamber.
  • the plate was placed in a 37 °C incubator (5% C0 2 , 86% RH) and cells were allowed to migrate for 4 hours.
  • the Transwell inserts were removed and discarded from the chamber after 4 hours migration.
  • the cells that migrated to the lower chamber were transferred to microcentrifuge tubes and were centrifuged for 5 min at 10,000 RPM. The supernatants were aspirated away, and the tubes frozen at -70 °C to facilitate cell lysis in the subsequent step.
  • cell lysis buffer/dye solution was prepared fresh by mixing CyQuant GR dye (400X stock) with IX lysis buffer. 200 ⁇ L ⁇ of the dye/lysis solution was added to the tubes containing the migrated cells and incubated 15 minutes at room temperature (dark); 150 ⁇ L ⁇ of the resulting cell lysates were transferred to a new 96 well plate and the fluorescence was read on a Molecular Dynamics SpectraMax M2 fluorimeter (ex 485, em 538).
  • control and Compound X treated cells showed only a modest decrease in migration when exposed to 10 ⁇ g/mL anti-CXCR4 antibody, but showed strong inhibition when treated with the CXCR4 antagonist AMD3100 (10 ⁇ g/mL).
  • Compound X had no effect on stem cell transwell migration toward SDF1, while AMD3100 inhibited stem cell migration substantially. Compound X presence did not interfere with the effect of AMD3100 or anti CXCR4 antibody. SDFla-CXCR4 axis is not involved in mobilizing effect of Compound X.
  • Streptozotocin was dissolved and diluted into citric acid buffer (pH 4.5) immediately before injections. Drinking water and food were removed for 6 hours from 8-week old C57BL/6 female mice (Charles River) before mice were given a single intraperitoneal dose of 150 mg/kg STZ (Day -4). Blood for glucose
  • STZ-induced diabetic mice received either one expanded bone marrow cell transplants on days +1 or three expanded bone marrow cell transplants on days +1, + 2 and +4.
  • one group of diabetic mice was treated with subcutaneous injections of 250 ⁇ g/kg of Compound X for 5 consecutive days for 3 weeks while Control group of mice received simultaneous Saline injections.
  • One group of diabetic mice was left untreated.
  • 8-week old C57BL/6 male mice (Charles River) were sacrificed and the cells removed from the femurs by flushing with Dulbecco's phosphate buffered saline (PBS).
  • PBS Dulbecco's phosphate buffered saline
  • the cells were pelleted by centrifugation and the cells were prepared for density centrifugation using a Percoll gradient. Low density fraction of cells was collected after centrifugation and resuspended in cIMDM containing 2 units/mL human erythropoietin, 100 ng/mL murine interleukin 3 and 100 ng/mL murine stem cell factor (all from R&D Systems) for ex vivo expansion. 2 ml aliquots of 4xl0 5 cells/mL were transferred to the wells of 24-well dishes in 2 mL. Cells were treated with Compound X (500 ng/mL,) at 24 and 72 hours.
  • Compound X 500 ng/mL,
  • FACS fluorescence activated cell sorting
  • IMDM complete IMDM (90% IMDM, 10% horse serum), split into four, 2mL aliquots and transferred to 12mL polypropylene round-bottomed tubes.
  • l0 ⁇ L ⁇ of CD38FITC, l0 ⁇ L ⁇ of CD49e/VLA-5PE and ⁇ , of CDl lbPerCP-Cy5.5 (all from Pharmingen) were added to each tube, the cells mixed and kept on ice, protected from light for 15 min.
  • Eight mL cIMDM was added, the cells pelleted by centrifugation for 5 min. and supernatant discarded.
  • cIMDM Cells were resuspended in lmL cIMDM (approximately 10xl0 6 cells/mL), passed through a 40mm filter (Falcon), transferred to 5ml FACS tubes and kept on ice protected from light until sorted.
  • Cells with a CD381o/VLA- 5 + /CDl lb " phenotype (also called 'R9 cells') were sorted into cIMDM, pelleted by centrifugation, resuspended in IMDM and a cell count using Trypan Blue (Sigma) exclusion for viability assessment performed on a hemacytometer. Three consecutive ex vivo expansions and R9 sorting were performed in order to transplant mice 3 times.
  • transplantation of R9 cells improved survival of diabetic mice after STZ injection.
  • Dose 150mg/kg used in this experiment provided longer survival of mice than previously used dose 160mg/kg.
  • Control mice injected with Saline for 3 weeks began to die while mice injected with stem cell mobilizing doses of Compound X and mice transplanted with R9 cells either one time or three times have survived 100%.
  • mice demonstrated substantial body weight loss: 50% of mice in control STZ group left without treatment and 50% mice in control group treated with Saline demonstrated substantial weight loss (about 20% of their initial body weight).
  • Such weight loss was registered only in 20% mice (2 out of 10) in groups transplanted with R9 cells 1 time or three times. No mice injected with stem cell mobilizing doses of Compound X had any substantial weight loss (data not shown).
  • Pancreatic insulin was measured by ECL assay as described above. In this model the control insulin levels in female C57B1/6 mice was found to be 5.768 + 0.12 ⁇ g/100mg tissue; this level usually dropped to 0.298 + 0.182 ⁇ g/100mg tissue one week after STZ injection. The results are presented in Figure 9.
  • EXAMPLE 6 Multiple subcutaneous injections of Compound X maintain an increased level of CFU-GEMM in peripheral blood of mice.
  • MethoCult 3434 media Stem Cell Technologies 03434. Briefly, peripheral blood from three mice was pooled, diluted (1 : 1, v/v) with 0.9% saline (APP Pharmaceutical) and layered (2: 1, v/v) onto 1- StepTM1.077 A (Accurate Chemicals AN224510) in 15-ml centrifuge tubes. Cells were centrifuged at 600 X g for 20 min. at room temperature.
  • the mononuclear cells were harvested from the interface between the plasma layer and the 1-StepTM A solution using a 1-ml syringe and 16G blunt-end needle (Stem Cell Technologies 28120). The cells were transferred to 15-ml centrifuge tubes and washed twice in IX PBS. The cells were placed into 35-mm Petri dishes at 100,000 cells per dish with 5 dishes per group. Cells incubated for 10 days in a 37°C and 5% C02 humidified incubator. CFU-GM colonies consisting of granulocytic and monocytic cells and CFU-GEMM colonies representing mixed cell populations of granulocytes, macrophages and erythroid cells were then scored using an inverted microscope.
  • Table 15 CFU-GEMM and CFU-GM number per ml of blood obtained from peripheral blood of mice on day 7 th after the first Compound X or vehicle injection.
  • Table 16 Flow cytometry analysis of primitive stem cells (Lin-kit+Sca+), self- renewing stem cells *CD150+CD48- and endothelial precursors (CD34+VEGF- R2/Flkl+) in spleens of mice on day 7 th after the first Compound X or vehicle injection.
  • HSCs hematopoietic stem cells
  • Compound X reversibly stimulates proliferation of HSC and by increasing their egress from bone marrow can bring into circulation double or triple that number of stem cells needed for regeneration without changing the cell composition in either the peripheral blood or the spleen. In a clinical setting this procedure could be repeated every 12hr generating multiple pulses of physiologically appropriate stem cells for potential tissue regeneration and the prevention of tissue damage associated with aging.
  • EXAMPLE 7 Multiple intravenous injections of Compound X induce mobilization of CFU-GEMM in diabetic db/db mice and accelerate wound repair.
  • Wound healing and tissue revascularization involves not only local repair processes but also support from bone marrow-derived repair cells.
  • a release of endogenous bone marrow progenitor cells was detected first in the peripheral blood then at the wound site in the early stages of wound repair.
  • the level of these released/mobilized progenitor cells in the peripheral blood was reduced in diabetic mice (Fiorina e.a.2010 and Tepper e.a.2010).
  • HSCs hematopoietic stem cells
  • Leptin receptor-deficient diabetic db/db mice are used routinely for studying diabetic wound healing and were selected for the present study.
  • Compound X subcutaneous administration was shown previously to increase efflux of multipotential colony forming progenitors, CFU-GEMM, in healthy mice. Therefore, this study was conducted in order to determine if Compound X could increase mobilization of CFU- GEMM into the circulation and accelerate wound healing in db/db diabetic mice.
  • Diabetic db/db - BKS.Cg-Dock7m +/+ Leprdb/J male mice Jackson Laboratories, stock # 000642) with verified blood glucose levels over 350mg/dL were used for mobilization experiments.
  • mice After methacrylate hardened, a transparent dressing (Tagaderm, Lab Safety Supply #53379) was placed over wounds. Two groups of mice (5 mice per group) received intravenous injections of either Vehicle or Compound X (5mg/kg) starting on day one post wounding and continuing daily for four consecutive days at Zeitgeber time 4 (ZT4), 4hr after onset of light to time mobilization of stem cells with circadian oscillations (Frenette et. al. 2008). The diabetic mice used in this and the following experiments had significantly elevated serum glucose level compared to aged matched C57B16 mice.
  • wounds were excised, fixed in 4% paraformaldehyde and processed for staining with rabbit anti-oc - smooth muscle actin antibodies and vascular density (number of blood vessels counted in the wound dermis area) compared. The number of blood vessels were counted on images as shown on Fig.12. Results are presented (Fig. 13).
  • mice received paired 6- mm full-thickness cutaneous wounds. Mice then received intravenous injections of either Vehicle or Compound X (5mg/kg) starting on day one post wounding and continuing daily for four consecutive days at ZT4. Wounds were monitored until complete closure in control group on day 21. To limit wound contraction during the healing process, a donut shaped silicone splint was centered on the wound and glued (cyanoacrylate adhesive) to the skin. Comparative analysis of the wound area measurements (Image J) was performed at day 14 post-wounding and is presented in Table 18.
  • mice were taken for the wound healing experiment when their glucose level was high; baseline glucose was measured by glucometer from tail bleed one day before the wound healing experiment next glucose measurement was taken at the end of the study, 7 weeks after the treatment with intravenous injections of Compound X or Saline. As shown (Fig. 14), 4 days treatment course with stem cell mobilizing agent Compound X not only accelerated wound healing process, but decreased glucose level in mice.
  • mice injected i.v. with 5mg/kg Compound X demonstrated substantially accelerated wound healing.
  • Wound closure in the Compound X treated group was mostly achieved by the 14 th day, while control wounds were closed between days 18-21 post- wounding.
  • Wound measurements are presented in Table 6 show that wounds were closed by day 14 in 11 of 14 mice treated with Compound X (95% - 100% closure) while mice treated with Saline had only 2 of 14 wounds closed at that time (Table 19; Fig. 15) .This was similar to data generated in previous experiments.
  • Table 19 Wound area measurements 14 days post- wounding.
  • Impaired wound healing is a major cause of increased morbidity and mortality in diabetic patients; the activity of Compound X in accelerating wound healing in diabetic mice supports its use in human wound healing, either alone or incombination with active local wound treatments including but not limited to PDGF and other growth factors, or grafts of natural or synthetic (cell-based) skin substitutes.
  • the reduction in blood glucose observed after treatment with intravenous Compound X indicates that mobilization of circulating progenitor cells supports pancreatic islet preservation or recovery in db/db mice, in addition to supporting wound healing.
  • Mobilization of bone-marrow-derived repair cells that maintain or support regeneration of pancreatic islets with Compound X is a new modality for treatment and prevention of diabetes.
  • EXAMPLE 8 Multiple intravenous injections of Compound X in diabetic db/db mice decreased the level of blood glucose. A study was conducted to determine whether treatment with Compound X could reduce the onset or severity of progressive severe type 2 diabetes using the db/db mouse model, which exhibits rapid onset of islet failure after an initial period of insulin resistance.
  • mice - BKS.Cg-Dock7m +/+ Leprdb/J (Jackson Laboratories, stock # 000642) become obese at 3 - 4 weeks of age. Elevation of plasma insulin usually occurs at 10 - 14 days and elevation of blood glucose at 6 - 8 weeks of age.
  • Compound X On the onset of hyperglycemia in db/db mice. Continuous Compound X treatments were performed during 5 weeks. At day 0, homozygous 7-week-old female db/db mice were divided into 2
  • mice received subcutaneous injections for 5 consecutive days: Group 1 received 0.9% saline and group 2 received 5 mg/kg Compound X. The following week mice received intravenous injections for 4 consecutive days. The next two weeks mice received subcutaneous injections 3 times per week and the last week of treatment was given intravenously 3 times per week.
  • Nonfasting blood glucose levels were measured weekly from tail bleeds with a handheld glucometer. Animal body weights were taken throughout the course of the study.
  • ACD3100 and CD26 modulate mobilization, engraftment, and survival of hematopoietic stem and progenitor cells mediated by the SDF-1/CXCL12- CXCR4 axis.” Ann N Y Acad Sci 1106: 1-19.
  • Hematopoietic stem cell yield depends on species-specific circadian timing. Cell Stem Cell; 3: 364-366.

Abstract

Cette invention concerne un procédé pour mobiliser les cellules souches à partir de la moelle osseuse par administration d'une quantité de Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gin-Val (SEQ ID No: 1) efficace pour mobiliser les cellules souches. Ce procédé est utile pour favoriser la conservation, la réparation, ou la régénération du tissu corporel, ou la revascularisation, chez un patient ayant besoin de ce traitement. En variante, les cellules souches mobilisées peuvent être collectées à des fins de greffe.
PCT/US2012/038610 2011-05-18 2012-05-18 Mobilisation des cellules souches, réparation et régénération tissulaire WO2012159044A1 (fr)

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JP6353073B2 (ja) 2013-12-20 2018-07-04 アドヴァンスド リジェン メディカル テクノロジーズ，エルエルシー 細胞回復のための組成物並びにその作製及び使用方法
US10772911B2 (en) * 2013-12-20 2020-09-15 Advanced ReGen Medical Technologies, LLC Cell free compositions for cellular restoration and methods of making and using same
JP6999575B2 (ja) 2016-04-29 2022-01-18 アドヴァンスド リジェン メディカル テクノロジーズ，エルエルシー マイクロｒｎａ組成物並びにその作製及び使用方法
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